A cohort study of Plasmodium falciparum infection dynamics in Western Kenya Highlands.

BACKGROUND: The Kenyan highlands were malaria-free before the 1910s, but a series of malaria epidemics have occurred in the highlands of western Kenya since the 1980s. Longitudinal studies of the genetic structure, complexity, infection dynamics, and duration of naturally acquired Plasmodium falciparum infections are needed to facilitate a comprehensive understanding of malaria epidemiology in the complex Kenyan highland eco-epidemiological systems where malaria recently expanded, as well as the evaluation of control measures. METHODS: We followed a cohort of 246 children residing in 3 villages at altitudes 1430 - 1580 m in western Kenya. Monthly parasitological surveys were undertaken for one year, yielding 866 P. falciparum isolates that were analyzed using 10 microsatellite markers. RESULTS: Infection complexity and genetic diversity were high (HE = 0.787-0.816), with ≥83% of infections harboring more than one parasite clone. Diversity remained high even during the low malaria transmission season. There was no significant difference between levels of genetic diversity and population structure between high and low transmission seasons. Infection turn-over rate was high, with the average infection duration of single parasite genotypes being 1.11 months, and the longest genotype persistence was 3 months. CONCLUSIONS: These data demonstrate that despite the relatively recent spread of malaria to the highlands, parasite populations seem to have stabilized with no evidence of bottlenecks between seasons, while the ability of residents to clear or control infections indicates presence of effective anti-plasmodial immune mechanisms.

Possible interruption of malaria transmission, highland Kenya, 2007-2008.

Highland areas where malaria transmission is unstable are targets for malaria elimination because transmission decreases to low levels during the dry season. In highland areas of Kipsamoite and Kapsisiywa, Kenya (population approximately 7,400 persons), annual household indoor residual spraying with a synthetic pyrethroid was performed starting in 2005, and artemether/lumefantrine was implemented as first-line malaria treatment in October 2006. During April 2007-March 2008, no microscopy-confirmed cases of malaria occurred at the sites. In 4 assessments of asymptomatic persons during May 2007-April 2008, a total of <0.3% of persons were positive for asexual Plasmodium falciparum by microscopy or PCR at any time, and none were positive by PCR at the last 2 sample collections. Our findings show that in such areas, interruption and eventual elimination of malaria transmission may be achievable with widespread annual indoor residual spraying of households and artemisinin combination therapy.

High prevalence of asymptomatic plasmodium falciparum infections in a highland area of western Kenya: a cohort study.

BACKGROUND: Transmission of malaria in an area of hypoendemicity in the highlands of western Kenya is not expected to lead to rapid acquisition of immunity to malaria. However, the subpopulation of individuals with asymptomatic Plasmodium falciparum infection may play a significant role as an infection reservoir and should be considered in malaria-control programs. Determination of the spatiotemporal dynamics of asymptomatic subpopulations provides an opportunity to estimate the epidemiological importance of this group to malaria transmission. METHODS: Monthly parasitological surveys were undertaken for a cohort of 246 schoolchildren over 12 months. The prevalence of P. falciparum infection among 2,611 blood samples was analyzed by both microscopy and polymerase chain reaction, and infection durations were determined. RESULTS: Infection prevalence and duration (range, 1-12 months) decreased with age and altitude. The prevalence was high among pooled blood samples recovered from children aged 5-9 years (34.4%) and from those aged 10-14 years (34.1%) but was significantly lower among blood samples obtained from older children (9.1%). The prevalence decreased from 52.4% among pooled blood samples from children living at an altitude of approximately 1,430 m to 23.3% among pooled samples from children living at an altitude of 1,580 m. CONCLUSIONS: The prevalence of asymptomatic P. falciparum infection was high, with polymerase chain reaction analysis detecting a significantly greater number of infections, compared with microscopy. Our results are consistent with gradual acquisition of immunity with increasing age upon repeated infection, and they also show that the risk of malaria transmission is highly heterogeneous in the highland area. The results provide strong support for targeted malaria-control interventions.

Molecular epidemiology of drug-resistant malaria in western Kenya highlands.

BACKGROUND: Since the late 1980s a series of malaria epidemics has occurred in western Kenya highlands. Among the possible factors that may contribute to the highland malaria epidemics, parasite resistance to antimalarials has not been well investigated. METHODS: Using parasites from highland and lowland areas of western Kenya, we examined key mutations associated with Plasmodium falciparum resistance to sulfadoxine - pyrimethamine and chloroquine, including dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps), chloroquine resistance transporter gene (pfcrt), and multi-drug resistance gene 1 (pfmdr1). RESULTS: We found that >70% of samples harbored 76T pfcrt mutations and over 80% of samples harbored quintuple mutations (51I/59R/108N pfdhfr and 437G/540E pfdhps) in both highland and lowland samples. Further, we did not detect significant difference in the frequencies of these mutations between symptomatic and asymptomatic malaria volunteers, and between highland and lowland samples. CONCLUSION: These findings suggest that drug resistance of malaria parasites in the highlands could be contributed by the mutations and their high frequencies as found in the lowland. The results are discussed in terms of the role of drug resistance as a driving force for malaria outbreaks in the highlands.

Microscopy underestimates the frequency of Plasmodium falciparum infection in symptomatic individuals in a low transmission highland area.

In an area with unstable malaria transmission, detection of Plasmodium falciparum infection in 379 symptomatic individuals was assessed by microscopy and three polymerase chain reaction (PCR) methodologies. P. falciparum infection was detected in 25% of patients by microscopy, 37% by nested PCR, 41% by merozoite surface protein-2 (MSP-2) PCR, and 45% by a ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Of the 64 individuals who were LDR-FMA positive, microscopy negative and did not receive treatment, 8 (12.5%) had persistent symptoms and returned for treatment. Malaria attributable fraction (MAF) in symptomatic individuals was 14.6% by microscopy (95% confidence interval [CI] = 6.6-21.8%) and 28.2% by nested PCR (95% CI = 17.9-37.2%). In this highland area, P. falciparum infection in symptomatic individuals is detected more frequently by PCR than microscopy, and most frequently by LDR-FMA. P. falciparum infection appears to resolve without treatment in most LDR-FMA-positive, microscopy-negative individuals, but is persistent in a subset of these individuals and requires treatment.

Plasmodium falciparum genetic diversity in western Kenya highlands.

The present study examined the genetic diversity and population structure of Plasmodium falciparum in western Kenya by analyzing the polymorphism of 12 microsatellite loci and two antigen loci. Malaria in highland areas is unstable and epidemic whereas malaria in lowland areas is endemic. Transmission intensity and malaria prevalence are substantially lower in the highlands than in the lowlands. Despite that the highland parasite populations exhibited reduced number of alleles, lower expected heterozygosity, and infection complexity in comparison to the surrounding lowland population, genetic diversity of the highland populations remained high in comparison to parasites from other meso-endemic regions. More than 70% of infections from western Kenya highland study sites were mixed genotype infections. Small but statistically significant genetic differentiation between highland and lowland populations was detected. These findings are discussed in the context of human travel and local transmission in the study area.

Amplified fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the yellow fever mosquito Aedes aegypti.

The yellow fever mosquito Aedes aegypti has been the subject of extensive genetic research due to its medical importance and the ease with which it can be manipulated in the laboratory. A molecular genetic linkage map was constructed using 148 amplified fragment length polymorphism (AFLP) and six single-strand conformation polymorphism (SSCP) markers. Eighteen AFLP primer combinations were used to genotype two reciprocal F2 segregating populations. Each primer combination generated an average of 8.2 AFLP markers eligible for linkage mapping. The length of the integrated map was 180.9 cM, giving an average marker resolution of 1.2 cM. Composite interval mapping revealed a total of six QTL significantly affecting Plasmodium susceptibility in the two reciprocal crosses of Ae. aegypti. Two common QTL on linkage group 2 were identified in both crosses that had similar effects on the phenotype, and four QTL were unique to each cross. In one cross, the four main QTL accounted for 64% of the total phenotypic variance, and digenic epistasis explained 11.8% of the variance. In the second cross, the four main QTL explained 66% of the variance, and digenic epistasis accounted for 16% of the variance. The actions of these QTL were either dominance or underdominance. Our results indicated that at least three new QTL were mapped on chromosomes 1 and 3. The polygenic nature of susceptibility to P. gallinaceum and epistasis are important factors for significant variation within or among mosquito strains. The new map provides additional information useful for further genetic investigation, such as identification of new genes and positional cloning.

Quantitative trait loci controlling refractoriness to Plasmodium falciparum in natural Anopheles gambiae mosquitoes from a malaria-endemic region in western Kenya.

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.

Fitness consequences of Anopheles gambiae population hybridization.

BACKGROUND: The use of transgenic mosquitoes with parasite inhibiting genes has been proposed as an integral strategy to control malaria transmission. However, release of exotic transgenic mosquitoes will bring in novel alleles along with parasite-inhibiting genes that may have unknown effects on native populations. Thus it is necessary to study the effects and dynamics of fitness traits in native mosquito populations in response to the introduction of novel genes. This study was designed to evaluate the dynamics of fitness traits in a simulation of introduction of novel alleles under laboratory conditions using two strains of Anopheles gambiae: Mbita strain from western Kenya and Ifakara strain from Tanzania. METHODS: The dynamics of fitness traits were evaluated under laboratory conditions using the two An. gambiae strains. These two geographically different strains were cross-bred and monitored for 20 generations to score fecundity, body size, blood-meal size, larval survival, and adult longevity, all of which are important determinants of the vector's potential in malaria transmission. Traits were analysed using pair-wise analysis of variance (ANOVA) for fecundity, body size, and blood-meal size while survival analysis was performed for larval survival and adult longevity. RESULTS: Fecundity and body size were significantly higher in the progeny up to the 20th generation compared to founder strains. Adult longevity had a significantly higher mean up to the 10th generation and average blood-meal size was significantly larger up to the 5th generation, indicating that hybrids fitness is enhanced over that of the founder strains. CONCLUSION: Hybridization of the two mosquito populations used in this study led to increased performance in the fitness traits studied. Given that the studied traits are important determinants of the vector's potential to transmit malaria, these results suggest the need to release genetically modified mosquitoes that have the same or very similar backgrounds to the native populations.

Mapping of chromosomal regions influencing immunological responses to gastrointestinal nematode infections in mice.

This paper reports the results of a genome-wide search for quantitative trait loci (QTL) influencing immunological responses to infection with the gastro-intestinal nematode parasite Heligmosomoides polygyrus in an F2 population created by crossing the resistant SWR and the susceptible CBA inbred mouse strains. Following infections, intestinal granuloma score at post mortem, mucosal mast cell protease 1, and IgE and IgG1 titres were recorded. The susceptible CBA mice had significantly higher IgG1, but significantly lower IgE, mucosal mast cell protease 1 and granuloma scores than SWR mice. Significant QTL were mapped to chromosomes 4, 11, 13 and 17 for granuloma score; chromosomes 12 and 17 for IgE; chromosome 10, 17 and 18 for IgG1 and chromosomes 1, 9, 10, 11, 17 and 18 for mucosal mast cell protease 1. Chromosomes 10, 11, 17 and 18 had QTL affecting more than one trait, and these are most likely to represent single QTL with multiple effects rather than multiple QTL. Some of these QTL map to regions known to harbour genes responsible for the induction of immunological responses to intestinal worms.

Chromosomal regions controlling resistance to gastro-intestinal nematode infections in mice.

Quantitative trait loci (QTL) associated with resistance to an intestinal worm in a well-defined murine model are described. These have been identified in an F(2) population derived from resistant (SWR) and susceptible (CBA) parental mouse strains infected with the gastro-intestinal nematode parasite Heligmosomoides polygyrus. Seven QTL located on six chromosomes are described, associated with components of the complex host response and the differential regulation of parasite survival and reproduction. The combined additive effects of the five significant QTL associated with worm survival (total worm count at necropsy) account for about 60% of the difference in worm count between the parental lines. The dominance effect for these five QTL are all in the direction of resistance, supporting the heterosis for resistance established from the mean worm count for the F(2) line relative to the parental lines. It is now possible to identify the comparative chromosomal regions of these QTL in livestock and humans and to consider the possibility of future improved control strategies. These may include breeding of resistant or tolerant livestock, development of vaccines, or identification of new anthelmintic drugs.